recupero di biofenoli dalle a.v. con tecniche di cromatografia
TRANSCRIPT
![Page 1: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/1.jpg)
Department of Industrial Chemistryand Materials
Recupero di biofenoli dalle A.V. contecniche di cromatografia
Leonardo Setti
w o r k s h o pAcque di vegetazione olearie:
trattamento e valorizzazione nel rispettodell’ambiente
Ottobre 23, 2007 – ENEA Centro Ricerche Casaccia
![Page 2: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/2.jpg)
Integrated valorization processIntegrated valorization process
RECOVERY CHEMICALS
BIO-TRASFORMATIONCHEMICALS
BIOFUELS
(Biogas, bioethanol, biodiesel,…)
COMPOSTING FERTILIZERS
WASTES
Step 1
Step 2
Step 3
![Page 3: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/3.jpg)
anthocyanins
Cross-Cross-sectional picture sectional picture of a green or black olive drupeof a green or black olive drupe
Phenolic acidsPhenolic acidsfor structuralfor structuralpolymerspolymers
? ? Phytoalexin secoiridoids oleuropeinPhytoalexin secoiridoids oleuropein
Source: Soler-Rivas et al. (2000) J. Sci. Food Agric. 80, 1013
![Page 4: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/4.jpg)
Olive oil
Olive oil productionOlive oil production
Olive oil millingwastes83%
17%
Husks and waste waters
4 x 106 ton/y in Italy
30 x 106 ton/y in theMediterranean area
![Page 5: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/5.jpg)
Ready for theextraction
Not Ready for theextraction
BiophenolsBiophenols in the Olive in the Olive milling wastesmilling wastes
4Verbascoside
Soluble Biophenols 1290-8760 mg/kg pulp of fresh olives
Soluble-Esterified Biophenols 570-2320 mg/kg pulp of fresh olives
Insoluble-bound Biophenols 1070-1620 mg/kg pulp of fresh olives
Xil
GalA
XilX ilX il X ilX ilX il
GalA GalA GalA
OO
OH
H3CO
OH
OCH 3
OO
Ga l
OH
OH
OO
GalAGalA
Glu
A
D
DB
C
GalA GalA GalA GalA GalAXil
GalA
XilX ilX il X ilX ilX il
GalA GalA GalA
OO
OH
H3CO
OH
OCH 3
OO
Ga l
OH
OH
OO
GalAGalA
Glu
A
D
DB
C
G alA G a lA G a lA G a lA G a lA
![Page 6: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/6.jpg)
Model of the Model of the primary cellprimary cell--wall wall of of dicots dicots (Carpita and (Carpita and GibeautGibeaut, 1993), 1993)
![Page 7: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/7.jpg)
Model of the Model of the primary cellprimary cell--wall wall of of dicotsdicots
Source: Saulnier and Thibault (1999) J.Sci.Food Agric. 79, 396 Guillon et al. (1989) Carbohydr. Res. 190, 97
![Page 8: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/8.jpg)
Galacturonic acid
arabinose
galactose
Ferulic acid
Rhamnose acid
Methyl-esterifiedgalacturonic acidAcetylatedgalacturonic acid
“smooth” region “hairy” region
Galactans
Arabinans
Diferulic acidbridge
Structure Structure of of sugar beet pectin sugar beet pectin ““hairyhairy”” regionregion
Source: Colquhoun et al. (1994) Carbohydr. Res. 263, 243
![Page 9: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/9.jpg)
CellCell--wall polysaccharide structure hydrolysiswall polysaccharide structure hydrolysisof of dicotsdicots
A Arabinase
B Arabinase
C Glucosidase
arabinosidase
D Arylesterase
Hydrolytic enzymaticactivities
Ara Ara Ara Ara Ara
Ara Ara Ara Ara Ara Ara
![Page 10: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/10.jpg)
Xil
GalA
XilX ilX il X ilX ilX il
GalA GalA GalA
OO
OH
H3CO
OH
OCH 3
OO
Ga l
OH
OH
OO
GalAGalA
Glu
A
D
DB
C
Ara Ara Ara Ara Ara
Ara Ara Ara Ara Ara Ara
Xil
GalA
XilX ilX il X ilX ilX il
GalA GalA GalA
OO
OH
H3CO
OH
OCH 3
OO
Ga l
OH
OH
OO
GalAGalA
Glu
A
D
DB
C
A ra A ra A ra A ra A ra
A ra A ra A ra A ra A ra A ra
MIXTURES OF VARIOUS ENZYMATIC ACTIVITIES MIXTURES OF VARIOUS ENZYMATIC ACTIVITIESFROM FROM AspergillusAspergillus niger niger and and TrichodermaTrichoderma longhibrachiatumlonghibrachiatum
![Page 11: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/11.jpg)
cellulasecellulase
pectinasepectinase
0.02 0.02 –– 0.1 0.1 µµmolmol/ml*/ml*minmin
![Page 12: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/12.jpg)
acqua+SANSA
0,0
5,0
10,0
15,0
20,0
0 1 2 3
Time (gg.)
Red
ucin
g S
ugar
(m
g/m
l)
Y
X
Z
CONTROL
HUSKS/WATER: 33% w/v
ENZYME CONC.: 1% v/v
pH: 4.88
Husks hydrolysisHusks hydrolysis
Husks + distilled water
The efficiency of the enzymatic hydrolysis was tested withthe husks of olive milling wastes
![Page 13: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/13.jpg)
HPLC/DAD HPLC/DAD analysis analysis on OMW on OMW before before andandafter the treatment after the treatment with with Y Y enzymaticenzymaticsolutionsolution
SEBPs HTy
IBBPs Phenolic acidsFerulic, caffeic, syringic,coumaric
+48% +98% +118% -17% +100%
g/l
![Page 14: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/14.jpg)
SEBPs HTy
glucosidase
esterase
D
E
OleuropeinGlucose
Elenolic acid
Hydroxytyrosol
![Page 15: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/15.jpg)
The extraction of these promising compounds hasThe extraction of these promising compounds hasstill some prominent problems to solve at anstill some prominent problems to solve at anindustrial scale:industrial scale:
1. the distant location and the recovery of wastes on the territory;2. the low concentration of bio-phenols in comparison to the large
amount of wastes to be treated;3. the management of a large amount of wastes to be transported to
the extraction plant and the relative costs;4. the large amount of wastes to be treated in time due to the scarce stability of
the wastes and their seasonality;5. the high-costs for the special disposal of the exhausted waste after solvent
extraction.
FoodProcessingwastes
Extractioncompany
Wastedisposal
![Page 16: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/16.jpg)
SHUTTLE PROJECTSHUTTLE PROJECT
Pifferi PG., Setti L., Bordado J.Plant for the treatment of waste watersEuropean Patent WO 03/004419 A1
European Project FAIR CT 97 3039European Project FAIR CT 97 3039““Natural antioxidants from olive millingNatural antioxidants from olive milling
processprocess””
![Page 17: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/17.jpg)
OMWW
Enzymatic treatment
Solid wastes Step 1
Enzymatic
solution
Step 2
Adsorption Step 3 Liquid wastes
Desorption Step 4
Washing of the resin
Liquid wastes
Resin
Step 6
Regeneration
Step 5
Finishing
Concentrated
liquid
Dry powder
Centrifugation
Scheme Scheme of theof theadsorptionadsorption//desorption processdesorption process
![Page 18: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/18.jpg)
OIL
LIQUIDWASTES
CONCENTRATEDANTIOXIDANT
EXTRACT
Solvent
Rigenerationsolvent
LIQUIDWASTES
CONCENTRATEDWASTES
OMWW
1 2 34
56
78
9
RESINS
Resins
Resins
Enzymaticsolution
SchemeScheme of the of the technological processtechnological process at a pilot at a pilotscale (1 mscale (1 m33/d) of olive /d) of olive milling waste watersmilling waste waters
Demonstrative plantat Tuyap Technologies (Istanbul, Turkey) (2002)
![Page 19: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/19.jpg)
Adsorption step
Sorbent: strong anionic exchanger styrene-divynil benzene copolymer
Sorbent concentration: 6% w/vStirred tank reactor at 25°C
![Page 20: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/20.jpg)
Desorption stepDesorption step
Sorbent concentration: 6% w/vSolvent: Ethanol 96% : HCl conc. (99:1)Stirred tank reactor at reflux of the solvent (~ 80°C)
Storage conditions for 68 d
![Page 21: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/21.jpg)
Regeneration cycles Regeneration cycles of of adsorptionadsorption//desorptiondesorptionprocess usingprocess using the the same resin same resin stockstock
![Page 22: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/22.jpg)
Yield of the pilot scale processYield of the pilot scale process
Recovery of Soluble Biophenols from OMWAverage adsorption rate: 54% +- 13% in 10 h timeAverage desorption rate: 78% +- 22% in 1 h timeAverage recovery: 41% +- 15%
Mass balanceFrom1 litre of OMW => 1.6 g of biophenols
0.8 g of Hydroxytyrosol
0.1 – 0.2 l of extract containing 8 – 16 g/l of biophenols and4-8 g/l of hydroxytyrosol
![Page 23: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/23.jpg)
OMWW25 m3/d
Enzymaticsolution
12.5 kg/d
OLIVE OIL180 – 450 kg/d
CONCENTRATEDWASTES
? kg/dResins
1500 - 3000kg/d
Solvent
Ethanol 49500 liters/dChloridric acid 500 liters/d
Biophenols115.5 kg/d
LIQUIDWASTES<25 m3/d
Ethanolicextract
50000 liters/d
CONCENTRATEDEXTRACT
2500 – 5000 liters/d
Biophenolsconcentration 6 -12 g/l
Biophenols amount63.2 kg/d
Ethanol45000 liters/d
Regeneration SolventChloridric acid liters/d
Water
LIQUIDWASTES<25 m3/d
1 2 3 4,5 6
78
9
SchemeScheme of the of the dimensioned technologicaldimensioned technologicalprocessprocess at at an industrialan industrial scale scale
![Page 24: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/24.jpg)
VITAFOODS - 5th VITAFOODS - 5th International Exhibition International Exhibition & & Conference Conference on on NutraceuticalsNutraceuticals and and Functional Foods Functional Foods - - Geneve Geneve 20022002
![Page 25: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/25.jpg)
““In vivoIn vivo”” ACTIVITIES OF OLIVE OIL WASTE WATER EXTRACTS ACTIVITIES OF OLIVE OIL WASTE WATER EXTRACTS RICH IN HYDROXYTYROSOL (HT)RICH IN HYDROXYTYROSOL (HT)
DeptDept. Of . Of Pharmacological SciencesPharmacological Sciences, , UnivUniv. Of . Of MilanMilanProf. Claudio GalliProf. Claudio Galli
•HT is bioavailable in animals and humans: it is dose-dependently adsorbed after oral ingestion
IN ANIMALS:
General antioxidant: increases the plasma antioxidant potential
Lipid antioxidant: reduces “in vivo” biomarkers of lipidperoxidation (urinary excretion of isoprostanes, non enzymaticoxidation products of arachidonic acid)
IN HUMANS:
Lipid antioxidant: increases urinary isoprostanes
Antithrombotic: reduces thromboxane formation
![Page 26: Recupero di biofenoli dalle A.V. con tecniche di cromatografia](https://reader034.vdocuments.mx/reader034/viewer/2022042710/6266cbc679d92266ab26e7ba/html5/thumbnails/26.jpg)
ConclusionsConclusions
1. Soluble biophenols can be extracted inquantitative way from agro-food wastes
2. Soluble biophenols were demonstrated to have animportant antioxidant powerful in vivo
3. Olive milling waste waters, winemaking wastesand mealmaking wastes could constitute a furtherindustrial resource of specific biophenols asnutraceutical compounds
4. The recovery/removal of the biophenols from thewastes could decrease the toxicity of the wastesthus improving any subsequent biological aerobicor anaerobic treatment